Download Increased concentration of metronidazole and its hydroxy metabolite

Pharmacological Reports
Copyright © 2012
2012, 64, 1276–1280
by Institute of Pharmacology
ISSN 1734-1140
Polish Academy of Sciences
Short communication
Increased concentration of metronidazole and its
hydroxy metabolite in colon cancer in women
Anna Sadowska1, Bogus³aw Kêdra2, Dariusz Cepowicz2, Lech
Rodziewicz3, Anna Fiedorowicz1, S³awomir Prokopiuk1, Wojciech Miltyk4,
Halina Car1
1
Department of Experimental Pharmacology, Medical University of Bialystok, Szpitalna 37, PL 15-295 Bia³ystok, Poland
2
2nd Department of General and Gastroenterological Surgery, Medical University of Bialystok, M. Sk³odowskiej-
Curie 24A, PL 15-276 Bia³ystok, Poland
3
Department of Hygiene Veterinary, Zwyciêstwa 26a, PL 15-959 Bia³ystok, Poland
4
Laboratory of Pharmaceutical Analysis, Medical University of Bialystok, Mickiewicza 2D, PL 15-089 Bia³ystok, Poland
Correspondence: Halina Car, e-mail: [email protected]
Abstract:
Background: Metronidazole (MTZ) is indicated in the prevention of infections during surgical procedures. However, some data
have shown that metronidazole has carcinogenic potential.
Methods: In the present work, we determined concentrations of metronidazole and its hydroxy metabolite (MTZOH) in colorectal
cancer patients. MTZ and MTZOH were measured in tumor tissue and surrounding healthy tissue by LC-ESI-MS-MS method.
Results: We found different concentration of MTZ and MTZOH in colorectal cancer and healthy tissue. Interestingly, we noted
a higher level of the above substances in women vs. men, both in healthy and cancerous gut.
Conclusion: We suggest that women are more exposed to a potential carcinogenic effect of metronidazole than men.
Key words:
metronidazole, MTZOH, colorectal cancer, human, women
Introduction
Colorectal cancer is the most common cancer diagnosed in Europe and also one of the leading causes of
cancer deaths – over 212,000 deaths in Europe in
2008 [5, 9, 11]. In most people, colorectal cancer develops slowly over several years. Before it develops,
a growth of tissue or tumor usually begins as a noncancerous polyp on the colon or rectum (cancer.org).
Another type of pre-cancerous stage is called dyspla-
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sia which over time can change into cancer. Dysplasia
occurs in people who have had ulcerative colitis or
Crohn’s disease for a long time [5]. These conditions
require treatment and the medications used have not
always been tested for carcinogenicity. The only effective treatment for this disease is surgery [3]. However, the tumor resection is associated with high risk
of perioperative infections.
Currently, the most important in the prevention of
infections is perioperative antibiotic prophylaxis
Metronidazole in colorectal cancer
Anna Sadowska et al.
which is significantly limiting the frequency of infections of surgical wounds [6]. There are many combinations of antibiotics and chemotherapeutic agents
used in surgical prophylaxis. In surgical patients, antimicrobial prophylaxis is indicated particularly in the
surgery of large intestine and rectum where anaerobic
bacteria predominate, particularly Bacteroides fragilis. Therefore, the most commonly used regimens are
cephalosporins and/or aminoglycosides and metronidazole. These are the medications which are widely
used in various diseases caused by microorganisms.
In addition to antimicrobial activity and on the basis
of their toxic potential, one can hypothesize that these
drugs may also affect healthy tissues as well as modify the tumor mass. Thus, the above substances need
further examination. It was shown that metronidazole
(MTZ), a chemotherapeutic agent, is genotoxic and
can lead to cancer formation in rodents [4, 7]. Metronidazole is an important drug for various infections
induced by anaerobic bacteria. Excellent penetration
into tissues, including central nervous system, bones
and joints, combined with bactericidal activity make
metronidazole very useful in the treatment of patients
with brain abscesses, skin infections and soft tissue,
bone and joint infections, or endocarditis. Although
patients before tumor resection receive metronidazole
or metronidazole along with other antibiotics as a normal rule, the influence of MTZ on both healthy and
cancerous tissues has not been described yet. It is also
not known whether the MTZ penetration in healthy
tissue differs from this of cancerously modified one
and whether drug absorption and its metabolism undergo any changes. Identification of the differences in
drug kinetics creates a new potential for grading of tumor tissue pathophysiology. These data could also indicate that the reconsideration of MTZ usage in patients suffering from colorectal cancer is a must.
Therefore, the aim of our study was to determine
the concentration of MTZ and its active hydroxy metabolite (MTZOH) in healthy colon tissue and tumor
revised.
Materials and Methods
Patients
The study was performed on colorectal resection
specimens removed from cancer and from healthy
tissue. The tissues were obtained from both women
aged 74 ± 8 years (n = 11) and men aged 63 ± 18
years (n = 8). Individuals were divided into 2 groups:
patients with metastases (N+) and without metastases
(N0) to lymph nodes. Colorectal cancer diagnosis was
based on clinical symptoms, colonoscopy and histopathological analysis of tumor and surrounding lymph
nodes taken during surgery. The patients revealed
T3N0 or T3N+ stage of tumor extent. T describes the
extent of spread through the colorectal layers (the
depth of the primary tumor), N estimates metastasis to
regional lymph nodes. The scale used for grading
colorectal cancers ranges from G1 to G4, dependently
on the differentiation status of the cancer tissue. All
patients were classified as G2 histological grade of
tissue differentiation.
Drug
All patients had been given 500 mg of the metronidazole iv 4 h prior to surgery.
Tissue samples
Samples of cancer and surrounding healthy colon
tissue were obtained about 4–5 h after administration
of MTZ. Tumor size (diameter) was approximately
the same in all patients (4.63 ± 1.5 cm).
LC-ESI-MS-MS method of MTZ and MTZOH
evaluation
Chemicals and reagents
Metronidazole (MTZ) as well as its hydroxy metabolite (MTZOH) and deuterated internal standard (IS)
hydroxymethylnitroimidazole d3 (HMMNI d3) were
obtained from Riedel-de-Haën (Fluka, Buchs, Switzerland). Acetonitrile, methanol LC-MS, methanol
LC, acetonitrile LC, acetone, formic acid (80–100%)
all analytical grades were purchased from Merck
(Darmstadt, Germany). Acetic acid (95.5%), sodium
sulfate, ammonia solution (25%) were obtained from
Baker (Deventer, The Netherlands). Strata SCX
(500 mg/3 ml) solid phase extraction (SPE) cartridges
were supplied by Phenomenex (Cat. No. 8B-SO10HBJ).
Standard solutions were prepared according to the
method described by Rodziewicz and Zawadzka [14].
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Sample preparation
Results and Discussion
The sample was weighed into glass tube and internal
standard (100 ng/ml of internal standard of HMMNId3, 100 µl) as well as acetonitrile (50 ml) were added
and vortex-mixed vigorously for 1 min. After adding
5 g of sodium sulfate, the sample was mixed and then
centrifuged at 3500 × g at temperature below 6°C for
10 min. The supernatant was transferred into a 50 ml
flask through 15 g of sodium sulfate and 5 ml of acetic acid was added. The extract was cleaned up on
SCX column. The column was conditioned with 5 ml
of 5% acetic acid in acetonitrile, washed with 5 ml of
acetone, 5 ml of methanol and 5 ml of acetonitrile,
and, finally, dried for 10 min. The nitroimidazoles
were then eluted with 5 ml of 5% ammonia in acetonitrile. The sample was evaporated to dryness under
a stream of nitrogen at 32°C.
Calibration curves
Calibration curves were constructed using analyte/internal standard peak area ratio vs. concentration of
analyte. The peak-area ratios of analyte to internal
standard were calculated using Analyst 1.4 Software.
LC-ESI-MS-MS
LC analyses were performed on a Luna C18(2) column (150 × 2 mm i.d., 3 µm) (Phenomenex, Torrance,
USA) using an Agilent 1100 series liquid chromatograph equipped with a binary pump and an autosampler (Agilent Technologies, Waldbronn, Germany).
MS analyses were carried out on an API 4000 triple
stage quadruple mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a turbo
ion spray interface. The instrument was operated in
Multiple Reaction Monitoring (MRM) mode using
the following transitions: m/z 188®123, 188®126
for MTZOH and m/z 161®143 for HMNNI d3 (IS)
d5, with collision energies (CE) of 20, 15 and 19 eV,
respectively.
Statistical analysis
The data are presented as the means ± SD. The statistical significance of the differences between the
means was determined by Student’s t-test; p < 0.05
was considered as the level of significance.
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The concentration of MTZ and MTZOH evaluated in
tumors equaled 3.86 ± 2.05 µg/g tissue and 0.54
± 0.66 µg/g tissue whereas in healthy tissue it was
2.97 ± 1.46 µg/g tissue and 0.49 ± 0.49 µg/g tissue,
respectively. The amount of MTZ and MTZOH in N+
cancer (2.16 ± 1.26 and 0.36 ± 0.44 µg/g tissue) was
slightly decreased in comparison with healthy tissue
(2.98 ± 1.59 and 0.41 ± 0.57 µg/g tissue). Oppositely,
both MTZ and MTZOH levels in N0 patients (4.39
± 3.88 and 0.85 ± 0.97 µg/g tissue) were elevated in
tumors vs. healthy colon (2.96 ± 1.54 and 0.63 ± 0.39 µg/g
tissue). A comparison of metastatic and non-metastatic carcinoma showed much higher concentrations
of MTZ and MTZOH (2-fold) in N0 vs. N+ tumor
tissue. Interestingly, cancers removed from women
presented considerably higher MTZ concentration
than those from men (Tab. 1). Similar results were
obtained in healthy tissue where MTZ level reached
significantly higher values for women in relation to
men. However, it should be noted that both men and
women tumor tissues revealed increased concentration compared with the healthy ones, but these differences were not statistically significant. The content of
MTZOH was higher in both healthy and tumor tissues
in female group when compared to male patients,
however, not reaching statistical significance.
We noticed the tendency to enhanced MTZ accumulation in both metastatic (3.81 ± 1.24 µg/g tissue)
and non-metastatic (6.04 ± 2.17 µg/g tissue) female
tumor tissues in comparison to men: 1.98 ± 1.18 µg/g
tissue (N+) and 3.08 ± 1.22 µg/g tissue (N0) which
did not gain statistical significance.
In the study, we found a differential (not statistically significant) concentration of MTZ in colorectal
cancer and surrounding healthy tissue, probably due
to greater absorption and accumulation of the chemotherapeutic in carcinoma vs. non-affected tissue. Absorption as well as accumulation of metronidazole
may depend on the tumor size and its morphological
structure. However, we noted approximately the same
tumor size in all patients. Larger necrotic tumors due
to increased hypoxia may absorb greater amounts of
the drug, which may explain increased (tendency)
concentration of MTZ in tumor.
Nevertheless, tumors presented distinct MTZ levels,
dependently on the metastatic status. Metastatic tumor (N+) revealed decreased (not significant) concen-
Metronidazole in colorectal cancer
Anna Sadowska et al.
Tab. 1. Concentration of MTZ and MTZOH (µg/g tissue) in tumor and healthy tissue of colorectal cancer patients
Men
Women
MTZ
MTZOH
MTZ
MTZOH
Tumor
2.534 ± 1.26
0.123 ± 0.05
4.826 ± 2.00*
0.794 ± 0.749
Healthy
1.62 ± 0.473
0.213 ± 0.07
3.786 ± 1.195*
0.658 ± 0.58
* p < 0.05 men (MTZ) vs. women (MTZ)
tration of the drug in comparison to non-metastatic
tissue (N0). Reduced drug absorption and decreased
accumulation in tumors of metastatic patients could
suggest that MTZ, apart from the primary tumor,
penetrates into lymph node metastatic tissues. Thus,
we hypothesize that pharmacokinetics is amended in
metastatic and non-metastatic cancer. In addition, we
noted higher content of measured MTZ in women vs.
men, both in healthy and cancerous gut. There still remains the question what determines the differences in
MTZ concentration between men and women, especially when considering patients’ age, with hormone
effects excluded.
These findings are important from a clinical point
of view as IARC classified MTZ as an animal carcinogen [13, 17]. The potential of MTZ to induce cancer has been under discussion since Rustia and Shubik
reported the induction of tumors in mice and rats by
this drug [15]. They observed a significant increase of
lung tumors and malignant lymphomas in female
Swiss mice. Female rats developed hepatomas and
mammary tumors while in males the number of pituitary and testicular tumors was elevated [2, 16]. Another study on animal carcinogenicity was performed
by Chacko and Bhide [7]. After MTZ intragastrical
administration to male and female Swiss mice, they
observed cystic ovaries, tumors of lung, spleen, liver
and thymus in a total of 13 females out of 40. In
treated males, 8 out of 38 developed tumors which included liver hemangiomas, lung and liver tumors [7].
On the basis of the experiments mentioned above, one
can easily assume that female animals developed tumor more often than male ones.
The data concerning MTZ human carcinogenicity are
still inconclusive. There have been few epidemiological
studies trying to evaluate the relationship between human cancer and the exposure to MTZ specifically. The
incidence of cervical cancer in women treated with
MTZ for vaginal trichomoniasis was found in epidemiological studies. However, trichomoniasis is a risk factor
for cervical cancer [1, 10]. Falagas et al. [8] determined
the influence of cancer after exposure to MTZ in comparison to patients that had not been exposed to the drug.
The main conclusion of their work was that the incidence (rate) of cancer among people who had been
given MTZ was almost the same as in the control group
[8]. However, there is a lack of a large cohort of people
treated with MTZ to clearly establish the risk that MTZ
treatment carries with itself.
The pharmacokinetics of MTZ in humans is not
completely established, but it is known that the drug
is metabolized mainly in the liver by cytochrome P450 system into hydroxy metabolites, acetic acid metabolites and glucuronide derivative. Hydroxy metabolite (MTZOH) and the parent drug are biologically active, where MTZOH has 30–60% of MTZ
biological activity. MTZOH has a longer half-life
(12 h) and contains nearly 50% of the antitrichomonal
activity of metronidazole [4, 12]. With reference to
the above data, we decided to examine the major metabolite of metronidazole. Our preliminary studies
showed that the level of MTZOH, dependently on sex
and metastases, displayed similar direction of changes
as MTZ, which suggests similar metabolism in tumor
and non-affected tissue.
Patients after resection of colon cancer receive
MTZ to avoid complications caused by infections.
Metronidazole and its metabolite MTZOH can be accumulated in tissues with low redox status and probably in metastatic cells. On the basis of the MTZ carcinogenic potential and the unknown MTZOH activity in tumor, it can be assumed that metronidazole
may enhance the invasiveness of cancer. The results
of our study may suggest that women are more exposed to such an effect of metronidazole than men.
Pharmacological Reports, 2012, 64,
1276–1280
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Acknowledgment:
This work was supported by the grant No. 3-10591L from the State
Committee for Scientific Research, Warszawa, Poland.
9.
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Received: November 24, 2011; in the revised form: April 18, 2012;
accepted: May 10, 2012.